Thousands of species of photosynthetic microbes are routinely cultivated at relatively small scale in the laboratory, in culture vessels ranging from several milliliters up to a few hundred liters in capacity. However, attempts to cultivate at larger scales, generally, necessary for commercial production, have proven successful for fewer than 10 species—despite a worldwide effort that has lasted half a century and consumed billions of dollars.
There are two basic types of culture vessels that have been employed in the effort to cultivate photosynthetic microbes at commercial scale: (1) Photobioreactors (Closed Systems), and (2) Open Systems.
(1) Closed Systems are characterized primarily by the provision of means to control access to the atmosphere. Gas exchange with the atmosphere is allowed to occur under controllable conditions. Carbon dioxide enters the culture vessel as a fuel for growth, and oxygen, the gaseous waste product of photosynthesis, is permitted to escape the culture vessel. (The carbon is assimilated into plant biomass, and the “dioxide”—oxygen—is expelled.) However, gas exchange occurs through filtration mechanisms that are designed to prohibit entry into the culture vessel of any species of photosynthetic microbe other than the one that is being preferentially cultivated therein.
Closed Systems are usually also designed to allow for the control of other environmental conditions. The provision for control of environmental variables such as temperature, pH, nutrient concentrations, and light makes it possible to optimize growth conditions for different species of microbial plants, which, like terrestrial plants, have distinct preferences for unique combinations of such variables.
For a given set of environmental conditions, all species of photosynthetic microbes grow at their maximum rate within a narrow range of cell concentrations. Accordingly, some Closed Systems are designed to operate as “turbidostats,” wherein the optical property of turbidity (opaqueness), which is a function of cell concentration, is monitored by means of programmable sensors that measure the optical density of the medium. The operator may specify a desired range of acceptable cell concentration, between a low value and a high value. The low value corresponds directly to a specific low optical density (the “low set point”), and the high value to a specific high optical density (the “high set point”). The optical density sensor is then programmed accordingly. When optical density attains a value that exceeds the designated upper set point, the turbidostat activates a control mechanism that provides for removing (harvesting) a fraction of the culture and replacing it with cell-free nutrient medium, thereby diluting the cell concentration to yield a value of optical density that matches the designated lower set point. The cells then will grow, increasing in concentration until optical density attains a value that once again exceeds the upper set point, at which time the cycle repeats.
Preferably a Closed System culture vessel is constructed primarily of transparent material, such as glass or plastic, that allows the transmission of photosynthetically active radiation (visible light), but which otherwise separates the culture medium from the atmosphere. Culture vessels may take many different shapes, but they all share in common one spatial dimension that limits their performance, and that is their depth relative to incident light intensity. This feature arises from a basic property of photosynthesis, namely, that photosynthetic rate is limited by light intensity. Thus, at any given light intensity, the rate of photosynthesis of a cell culture is maximized as a function of the Lighted Area, i.e. the area of the culture medium that is exposed to light (not necessarily the Surface Area, which may include areas of the culture vessel that are not exposed to light). Consider two culture vessels, both outdoors and exposed to sunlight. One is in the shape of a rectangular pond with solid sides and bottom, and the second is in the shape of a transparent cylinder, placed horizontally on top of the ground. For the rectangular pond the Surface Area includes the top, bottom, and sides of the pond, but only the top area of the culture medium is exposed to sunlight. For such an outdoor pond, then, the Lighted Area is equal to less than half the Surface Area. By comparison, for the transparent cylinder, the Surface Area is the entire surface of the cylinder and, no matter what the time of day, half of the Surface Area will always be directly illuminated by sunlight. Thus, for the cylinder, the Lighted Area is equal to half the Surface Area
A second factor affecting the relationship between photosynthesis and light is the cell concentration in the culture medium. The greater the cell concentration within a medium, the less the depth to which light may penetrate, because light penetration decreases approximately exponentially as a function of cell concentration. In other words, if the cell concentration increases at a constant rate, light disappears faster and faster. At some depth in a cell culture, then, light will actually decrease to zero.
As a practical matter, the optimal culture depth for photosynthetic microbes exposed to full sunlight is generally in the range of 10 to 20 centimeters. No advantage can be gained by providing greater depth of the culture, because the concentration of cells per unit Lighted Area will remain the same, and deeper cells will not receive enough light. The optimal depth, then, puts a limit on the normal operating capacity of any culture system, regardless of its Lighted Area. This phenomenon is a critically important feature in the design of cultivation systems. Greater volumes require more, materials, at greater cost, but at some point the increase in volume provides no increase in productivity per unit Lighted Area.
Cultures of photosynthetic microbes generally require stirring or mixing in order to maintain a homogeneous distribution of cells in the medium. The natural tendency of photosynthetic microbes in still water is to form dense aggregations, within which the properties of the medium are altered to the detriment of the culture. On a microscale, within the aggregation, the availability of light and the concentration of nutrients and gases become so different from the remainder of the medium that growth is limited. Some species have appendages known as cilia or flagellae that allow them to swim; such motile (“moving”) species actively form aggregations. Most non-motile species are heavier than water and will sink, forming a passive aggregation on the bottom. To prevent such aggregations, Closed Systems must provide a means for creating turbulence using devices such as airlifts or pumps.
(2) Open Systems differ from Closed Systems in one critical feature, namely that they are open to the atmosphere. This feature is advantageous to both construction and operation, in several ways. First, because the Lighted Area of an Open System is exposed directly to sunlight, there is no requirement to use a transparent material to construct the culture vessel; this affords broad latitude in the choice of materials. Second, because no material is used to cover the Lighted Area of the Open System, the amount and cost of material is reduced by about half. Third, Open Systems are generally easier to clean than Closed Systems. Over time the inner surface of any culture vessel will tend to accumulate a film of microbial growth. In a Closed System the accumulation of such a film on the Lighted Area will absorb light; the consequent decrease in light intensity causes a decrease in productivity. In both Open Systems and Closed Systems the culture vessel surface can accumulate microbial films of undesirable species that may be detrimental to growth and production of the desired species. In either case, the culture vessel surface will require cleaning from time to time. As a practical matter, Open Systems allow a much wider choice of cleaning methodologies. For example, people and large types of mechanical cleaning equipment such as hoses, pressure washers; and scrubbers that cannot enter the confined space of a Closed System can easily enter an Open System.
The principal disadvantage of an Open System is that, by being open to the atmosphere, it is susceptible to contamination by unwanted species. One may begin the operation of an Open System culture with only one desired species of photosynthetic microbe. However, undesired species will inevitably be introduced, whether by atmospheric transport or other means. Any undesired species that grows faster than the desired species in the same environmental conditions will, over time, outcompete the desired species and will ultimately dominate the culture.
In summary, Closed Systems are designed specifically to prohibit contamination by undesired species, with the expectation that continuous cultivation of a desired species may be possible for a much longer period than would be possible in an Open System. However, Closed Systems are more complicated to construct and operate. Open Systems afford a wider choice of materials for construction, and also afford a wider choice of cleaning methodologies. Closed Systems require additional operating practices, such as the use of sterile technique during fluid transfers, which call for greater time and expertise on the part of the operator.
Theoretical differences between Closed Systems and Open Systems have been borne out in practice. The first photosynthetic microbe was isolated from nature and grown in pure culture little more than a hundred years ago, but it was not until the late 1930s that sufficiently large volumes of a single species could be cultivated to permit chemical analysis. By the 1940s various species were being grown in laboratory cultures of about 25 liters, and it was discovered that, by altering environmental conditions of the culture, either the oil or protein content of some species could be made to exceed 60% of the total cell mass.
The first attempts at large-scale cultivation began in the 1950s, stimulated by widespread interest in photosynthetic microbes as a source of cheap protein for foods and animal feeds. The first Open Systems, built in Germany, took the shape of shallow, elongated, recirculating raceways, with flow provided by a paddlewheel device. Nationally-funded programs developed rapidly throughout the world, all following the German “open pond” design. The first open ponds had capacities of just a few thousand liters. By the late 1950s, capacities of almost 100,000 liters had been attained and, by the late 1960s, almost 1,000,000 liters. Such increases in capacity brought economies of scale.
Hundreds of species were tested in the laboratory, and attempts were made to grow the best protein producers in open ponds during the 1960s and 1970s. Only a few species proved to be amenable to sustained cultivation. These few species, such as Spirulina platensis and Dunaliella salina, went on to become the basis of commercial production, effected in open pond systems covering hundreds of acres. The successful commercial species proved to be “extremophiles,” which thrive in conditions of unusually high pH or salinity. Most species prefer conditions that prevail in nature, where numerous species thrive simultaneously. For two decades, all attempts to cultivate single-species cultures of non-extremophiles in open ponds failed after less than a few months because they were contaminated by other species that thrived under the same environmental conditions.
Renewed interest in large-scale cultivation was stimulated in the 1980s and 1990s by the prospect of producing renewable biofuels using oils from photosynthetic microbes as a feedstock. During this period government agencies of the USA and Japan, for example, invested approximately $150 million in such an effort. Such programs shared two goals: first, to collect and identify species of photosynthetic microbes that produce high concentrations of oil and then to determine the environmental conditions under which they do so; and, second, to design and demonstrate the operation of large-scale cultivation systems for the production of biofuel feedstocks using species that had been developed in the laboratory. Both programs succeeded at the first goal, but failed at the second.
Laboratory studies quantified earlier findings. Culture collections of hundreds of species were amassed. Research on numerous strains demonstrated that, in general, nitrogen sufficiency (nitrogen is needed for protein synthesis) promoted high growth rates and low oil content, whereas nitrogen deficiency resulted in low growth rates and high oil content. For some species, it has also been noted that stress, caused by factors such as high light intensity or very high temperatures, can induce species to shift from protein synthesis to oil synthesis. Species capable of optimal oil production—the highest oil content at the highest growth rate—were selected for large-scale production trials.
Large-scale production was once again attempted in the late 1980s and early 1990s using open pond systems. Operating results were similar to those obtained for the three previous decades. Promising oil-producing species were selected from the collections, and cultures were inoculated into the ponds. However, as in prior experience, single-species cultures could not be maintained for more than a few weeks or months. The final report of the US program referred to this phenomenon as an “uncertainty with the nature of species control achieved.”
By the 1990s the status of large-scale cultivation had not progressed beyond the point reached in the 1960s. Three types of microalgae—Spirulina, Dunaliella and Chlorella were being cultivated at facilities using open pond systems covering more than 100 acres. Scores of other species had been attempted worldwide, but all attempts had failed. The biofuels programs, in particular, had been unable to grow any desired species at any scale outside the laboratory. Moreover, the biofuels programs had focused on attempts to demonstrate the highest possible biomass production rates under nutrient-sufficiency, conditions that are known from laboratory studies to favor low oil content. No attempts were made at large-scale to maximize oil production.
Large scale Closed System technology began to receive significant attention in the early 1990s, once it became evident that cultures of most species exposed to atmosphere were not sustainable. At that time, the largest Closed Systems that had ever been used were no more than a few thousand liters in capacity. Advances in the past decade have succeeded at increasing reactor capacity by a factor of about 10, to about 30,000 liters. But this is nowhere near the rate of increase achieved for Open System capacity that, also over a decade (in the 1950s to 1960s), increased by a factor of 1,000.
The upper limit of Closed System capacity is, in large part, a direct consequence of inherent design requirements. All basic Closed System designs in use today were first developed in the 1950s, and may be categorized as follows: (1) vertical bags, tubes, or towers; (2) flat-plate reactors; and (3) horizontal tubes. Vertical systems are constrained by height limitations. Even when exposed to full sunlight, most cultures achieve such high cell densities that light is almost entirely absorbed at a distance of more than 15 to 20 cm from the Lighted Area. This constraint limits the diameter of the culture vessel to no more than 30 or 40 cm. To achieve a capacity of more than 10,000 liters, for example, a 40-cm diameter vertical system would have to be more than 80 meters (260 feet) high. Such dimensions present clear challenges in structural engineering which, even if achievable, become increasingly complex the greater the volume of the system. One of the obvious solutions has been to introduce an illumination system within the reactor, but experience has shown that this introduces other problems, of which bio-fouling may be the greatest. Over a relatively short time, the surface of the light source tends to become covered with a microbial film, sharply reducing light intensity and thus defeating the purpose of the light source. Removing the culture and cleaning the vessel is one option, but hardly desirable if the goal is sustained operation. Another common anti-fouling option, making the surface of the light source toxic to microbes, is clearly undesirable. In general, the use of internal illumination makes the system more complex.
Horizontal systems such as flat-plate reactors and horizontal tubes eliminate the need for the structural engineering required of vertical systems. Using the earth's surface for structural support, the potential capacity of such systems might appear limitless. However, the capacity of horizontal systems is generally limited by the requirement for turbulent flow, whether used to maintain adequate mixing or to fill and empty the culture vessel.
Turbulent flow in a pipe or a channel is described by the Reynolds number, defined as the velocity of the fluid multiplied by the “characteristic length” of the pipe or channel, and divided by the viscosity of the fluid. The Reynolds number does not have any units, like inches or pounds, and is therefore “dimensionless,” like “one-half” or “two-thirds”. The characteristic length of a fluid-filled pipe is its diameter; the characteristic length of a wide channel is its depth. For a fluid of constant viscosity, flow will become increasingly turbulent as the velocity of flow increases. Turbulence also increases in proportion to the characteristic length; this happens because pipe and channel surfaces are “sticky.” Surfaces cause friction that slows down the flow; the flow rate is almost zero next to the surface, and increases with distance away from the surface. Thus, in a pipe or channel with small characteristic length, the surface friction will have a great effect on the average flow. By contrast, in a pipe or channel with large characteristic length, the surface friction will have little effect on the average flow, and turbulence will be greater.
Surface friction also adds up over distance. Imagine a very long pipe through which water is propelled by a pump. At the origin, near the pump, the flow is turbulent. The farther the fluid moves down the pipe, the more surface it is exposed to and, the more surface it is exposed to, the more its flow is slowed by friction. At some point from the origin, the accumulated friction has removed so much energy from the fluid flow that it ceases to be turbulent. This happens when, the Reynolds number falls below a value of about 2000, and then the flow is said to be “laminar.”
Laminar flow is not desirable in cell cultures because in such conditions the cells have a tendency to aggregate, either by sinking or swimming. Turbulent flow prevents such aggregations. Imagine, for example, how sand particles would rapidly sink to the bottom in a still pond, but would not do so in a large breaking wave or a rapidly moving stream.
In summary, then, turbulent flow is maintained by avoiding very low fluid velocities, very small characteristic lengths, and very long channels. The characteristic length for horizontal Closed Systems such as flat-plate reactors or horizontal tubes is the depth of the culture, which, as explained previously, has a practical upper limit of about 20 cm. One can create turbulent flow in a flat-plate reactor or a horizontal tube with any number of devices such as pumps or airlifts. However, with increasing distance from the origin of the flow, turbulent energy is lost to friction such that, at some finite distance flow becomes laminar. In laminar flow conditions the cells of most photosynthetic microbes will sink to the bottom of the reactor. This is undesirable for many reasons, not the least of which is that harvesting the cells becomes problematical. One solution is to provide more turbulent energy at the source, but this is acceptable only to an upper limit where mechanical shear damages the cells themselves. Yet another solution is to provide multiple pumps, for example, throughout the reactor, but this approach introduces additional complexities of both construction and operation.
As a matter of practice, vertical Closed Systems are limited to a capacity of less than about 1,000 liters, and horizontal Closed Systems appear to be limited to capacities less than about 50,000 liters. For the purpose of large-scale cultivation of photosynthetic microbes, Closed Systems are much more costly and complex to construct and operate than Open Systems. This is because each independent system requires its own independent infrastructure: a set of devices or mechanisms for providing turbulent mixing, introduction and removal of medium, and monitoring and control of variables such as pH and temperature. To cover a given area of land with Closed Systems requires at least 10 times more infrastructure than covering the same area of land with Open Systems, rendering Closed System cultivation much more complicated.
In practice, every cultivation system for photosynthetic microbes involves a coupling of both Open Systems and Closed Systems at some scale. All cultivation systems, regardless of scale, ultimately depend for their original inoculum of cells on culture collections routinely maintained around the world. All culture collections exclusively maintain their cell cultures in Petri dishes, test tubes, or sterilized flasks—all of which are, strictly speaking, Closed Systems. Even large-scale production systems that might be considered to consist “purely” of Open Systems must rely ultimately on a Closed System to supply the original inoculum.
The main technical conundrum for the production of photosynthetic microbes is that Open System technology has advanced to a large scale that is economical and relatively easy to operate, but cannot provide sustainable production of desired microbes. By contrast, Closed Systems do provide sustainable production of desired microbes, but even at their largest scale they are costly and complicated to operate.
Thus, there is a need for a production method that provides for sustainable production by reducing the potential for contamination and yet does not substantially increase the complexity or cost of construction or operation.
It is therefore an object of this invention to provide an effective method for sustainable production of photosynthetic microbes at large scales that may be easily constructed and does not increase the complexity or cost of construction or operation.
It is a still further object of this invention to provide a method of production that is especially suited to optimizing the production of oils and other useful products from photosynthetic microbes. Oils and other useful products may then be extracted and purified from the aggregate biomass by means of a variety of chemical methods.